Glutamine PRPP amidotransferase catalyzes the initial reaction in de novo purine nucleotide synthesis and is the key regulatory enzyme in the pathway. Genes encoding glutamine PRPP amidotransferase have been cloned from more than 20 organisms including bacteria, eukarya, and archea. In particular, the enzymes from Escherichia coli and Bacillus subtilis have been purified to homogeneity and are well-characterized, including having the X-ray structures determined for these enzyme species. The E. coli and B. subtilis enzymes are both homotetramers and are representative of two classes of glutamine PRPP amidotransferases. Enzymes of the B. subtilis class are synthesized with an NH.sub.2 terminal propeptide and an Fe--S center, whereas enzymes of the E. coli class have neither.
Purine nucleotide biosynthesis is regulated, at both the gene (transcription/translation) and the protein (enzymatic activity) level. Attempts at increasing the biosynthesis of purine nucleotides have focused on the deregulation of genes encoding pathway enzymes. However, the enzymatic activity of a key pathway enzyme, glutamine PRPP amidotransferase, is inhibited by the pathway end-products, adenine and guanine nucleotides. Therefore an effective strategy for enhancing host cell production of purine nucleotides includes the use of a modified glutamine PRPP amidotransferase, wherein the modification reduces the sensitivity of the enzyme to end-product inhibition by adenine and guanine nucleotides.
The two classes of glutamine PRPP amidotransferase enzymes (as represented by the E. coli and B. subtilis enzymes) exhibit different end-product inhibitory patterns by adenine and guanine nucleotides. GMP is the single strongest inhibitor of E. coli glutamine PRPP amidotransferase, and AMP is the strongest inhibitor of the B. subtilis enzyme. Notwithstanding this difference, there is one common distinctive characteristic of the inhibition by nucleotides. Certain pairs of adenine and guanine nucleotides give more than additive inhibition compared to the individual nucleotides, a phenomenon called synergistic inhibition. AMP plus GMP has been reported to be a strong synergistic nucleotide pair for the E. coli enzyme, although more recent experiments suggest that GDP plus AMP may be the most effective synergistic pair. The strongest synergistic pair for the B. subtilis enzyme has been reported to be ADP plus GMP.
The synergistic inhibition of glutamine PRPP amidotransferase implies the existence of separate binding sites for adenine and guanine nucleotides on each subunit. X-ray structures of B subtilis and E. coli enzymes have identified four nucleotide binding sites per half-tetramer, two equivalent allosteric A sites between subunits, each with an adjacent catalytic C sites. It has been reported that synergistic binding of GMP to the A site and AMP to the C site could account for the synergistic inhibition of the E. coli enzyme.
A more systematic study of the mechanism for synergistic inhibition of the B. subtilis glutamine PRPP amidotransferase, as reported herein, confirms that ADP and GMP are the most synergistic pair and demonstrates that synergistic inhibition results from synergistic binding. An X-ray structure of a ternary enzyme ADP-GMP complex establishes that ADP binds to the A site and GMP to the C site and that synergism results from a specific interaction between the .beta.-phosphate of a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. These results establish the mechanistic basis for synergism.